Insertion of microRNA targets into the flavivirus genome alters its highly neurovirulent phenotype

Flaviviruses such as West Nile, Japanese encephalitis, and tick-borne encephalitis (TBEV) viruses are important neurotropic human pathogens, causing a devastating and often fatal neuroinfection. Here, we demonstrate that incorporation into the viral genome of a target sequence for cellular microRNAs expressed in the central nervous system (CNS) enables alteration of the neurovirulence of the virus and control of the neuropathogenesis of flavivirus infection. As a model virus for this type of modification, we used a neurovirulent chimeric tick-borne encephalitis/dengue virus (TBEV/DEN4) that contained the structural protein genes of a highly pathogenic TBEV. The inclusion of just a single target copy for a brain tissue-expressed mir-9, mir-124a, mir-128a, mir-218, or let-7c microRNA into the TBEV/DEN4 genome was sufficient to prevent the development of otherwise lethal encephalitis in mice infected intracerebrally with a large dose of virus. Viruses bearing a complementary target for mir-9 or mir-124a were highly restricted in replication in primary neuronal cells, had limited access into the CNS of immunodeficient mice, and retained the ability to induce a strong humoral immune response in monkeys. This work suggests that microRNA targeting to control flavivirus tissue tropism and pathogenesis might represent a rational approach for virus attenuation and vaccine development.     

黄病毒属病毒E蛋白结构和功能研究进展

黄病毒属病毒是单股正链RNA病毒,病毒基因组编码至少三种结构蛋白(衣壳蛋白C、膜蛋白M和包膜蛋白E)和七种非结构蛋白。其中E蛋白是病毒的重要抗原成分,包含有中和抗原表位和型特异性抗原表位,决定了病毒的细胞嗜性和毒力,与病毒的吸附、穿入、致病等作用密切相关,并且具有血凝活性,能刺激机体产生中和抗体和血凝抑制抗体。研究E蛋白的结构与功能对于深入了解黄病毒致病机制和免疫应答特点,研发疫苗和特异性抗病毒药物均有重要的指导意义。为此,综述了近年来黄病毒E蛋白有关结构与功能的研究进展及其生物学意义。     

Lipids and flaviviruses,present and future perspectives for the control of dengue,Zika, and West Nile viruses

Flaviviruses are emerging arthropod-borne pathogens that cause life-threatening diseases such as yellow fever, dengue, West Nile encephalitis, tick-borne encephalitis, Kyasanur Forest disease, tick-borne encephalitis, or Zika disease. This viral genus groups > 50 viral species of small enveloped plus strand RNA virus that are phylogenetically closely related to hepatitis C virus. Importantly, the flavivirus life cycle is intimately associated to host cell lipids. Along this line, flaviviruses rearrange intracellular membranes from the endoplasmic-reticulum of the infected cells to develop adequate platforms for viral replication and particle biogenesis. Moreover, flaviviruses dramatically orchestrate a profound reorganization of the host cell lipid metabolism to create a favorable environment for viral multiplication. Consistently, recent work has shown the importance of specific lipid classes in flavivirus infections. For instances, fatty acid synthesis is linked to viral replication, phosphatidylserine and phosphatidylethanolamine are involved on the entry of flaviviruses, sphingolipids (ceramide and sphingomyelin) play a key role on virus assembly and pathogenesis, and cholesterol is essential for innate immunity evasion in flavivirus-infected cells. Here, we revise the current knowledge on the interactions of the flaviviruses with the cellular lipid metabolism to identify potential targets for future antiviral development aimed to combat these relevant health-threatening pathogens.     

Traditional and novel approaches to flavivirus vaccines

Yellow fever, dengue, Japanese encephalitis and tick-borne encephalitis viruses are the medically most important members of the Flavivirus genus composed primarily of arboviruses. In this paper, we review the commercially available traditional flavivirus vaccines against yellow fever, Japanese encephalitis, and tick-borne encephalitis, as well as modern approaches to flavivirus vaccines. Formalin inactivation technology has been employed to produce killed vaccines. Flaviviruses have been attenuated by multiple passages in animal tissues and cell cultures to produce empirical live attenuated vaccines. The use of traditional methods is being pursued to develop vaccines against other flavivirus diseases, such as dengue, and to improve existing vaccines, such as for Japanese encephalitis. With the recent development of infectious clones, rational approaches to attenuated flavivirus vaccines have employed the introduction of specific mutations into wild type viruses and chimerisation between different viruses. Novel methods for delivery of live vaccines, such as inoculation of infectious DNA or RNA, have been described. Other approaches, such as the construction of protein subunit, expression vector-based and naked DNA vaccines, have been proposed to create alternate vaccine candidates.     

细胞焦亡在黄病毒致病机制中的作用

黄病毒(flavivirus)是一类具有包膜的单股正链RNA病毒,经蚊虫叮咬传播,是新发突发传染性疾病的重要病原体,严重威胁着人类健康。尽管不同黄病毒引起的临床疾病不同,但是它们的临床症状却有一些相似之处,发热是黄病毒感染后最常见的症状,而且往往表现为高热。研究发现,寨卡病毒和日本脑炎病毒感染中存在胱天蛋白酶1 (caspase1)依赖的炎性反应,而这一过程与细胞焦亡(pyroptosis)的部分机制相吻合。细胞焦亡是一种依赖于胱天蛋白酶(caspases)的炎性细胞程序性死亡类型,其特征有焦孔素(gasdermin)介导的孔形成、细胞肿胀破裂和炎性细胞因子释放。本文对黄病毒感染引起的固有免疫中巨噬细胞的焦亡现象进行综述,对细胞焦亡的分子机制、细胞焦亡的重要组分作用进行总结,分析了细胞焦亡与代表性黄病毒之间的关系,以期为细胞焦亡在黄病毒致病机制的后续研究提供参考,为抗病毒感染治疗提供新的思路。     

Comparative neuropathogenesis and neurovirulence of attenuated flaviviruses in nonhuman primates

Based on previous preclinical evaluation in mice and monkeys, the chimeric TBEV/DEN4Delta30 virus, carrying the prM and E protein genes from a highly virulent Far Eastern strain of tick-borne encephalitis virus (TBEV) on the backbone of a nonneuroinvasive dengue type 4 virus (DEN4), has been identified as a promising live attenuated virus vaccine candidate against disease caused by TBEV. However, prior to use of this vaccine candidate in humans, its neurovirulence in nonhuman primates needed to be evaluated. In the present study, we compared the neuropathogeneses of the chimeric TBEV/DEN4Delta30 virus; Langat virus (LGTV), a former live TBEV vaccine; and yellow fever 17D virus vaccine (YF 17D) in rhesus monkeys inoculated intracerebrally. TBEV/DEN4Delta30 and YF 17D demonstrated remarkably similar spatiotemporal profiles of virus replication and virus-associated histopathology in the central nervous system (CNS) that were high in cerebral hemispheres but progressively decreased toward the spinal cord. In contrast, the neurovirulence of LGTV exhibited the reverse profile, progressing from the site of inoculation toward the cerebellum and spinal cord. Analysis of the spatiotemporal distribution of viral antigens in the CNS of monkeys revealed a prominent neurotropism associated with all three attenuated viruses. Nevertheless, TBEV/DEN4Delta30 virus exhibited higher neurovirulence in monkeys than either LGTV or YF 17D, suggesting insufficient attenuation. These results provide insight into the neuropathogenesis associated with attenuated flaviviruses that may guide the design of safe vaccines.     

Structural basis for the preferential recognition of immature flaviviruses by a fusion‐loop antibody

Flaviviruses are a group of human pathogens causing severe encephalitic or hemorrhagic diseases that include West Nile, dengue and yellow fever viruses. Here, using X‐ray crystallography we have defined the structure of the flavivirus cross‐reactive antibody E53 that engages the highly conserved fusion loop of the West Nile virus envelope glycoprotein. Using cryo‐electron microscopy, we also determined that E53 Fab binds preferentially to spikes in noninfectious, immature flavivirions but is unable to bind significantly to mature virions, consistent with the limited solvent exposure of the epitope. We conclude that the neutralizing impact of E53 and likely similar fusion‐loop‐specific antibodies depends on its binding to the frequently observed immature component of flavivirus particles. Our results elucidate how fusion‐loop antibodies, which comprise a significant fraction of the humoral response against flaviviruses, can function to control infection without appreciably recognizing mature virions. As these highly cross‐reactive antibodies are often weakly neutralizing they also may contribute to antibody‐dependent enhancement and flavi virus pathogenesis thereby complicating development of safe and effective vaccines.     

The Chemokine Receptor CCR5, a Therapeutic Target for HIV/AIDS Antagonists,Is Critical for Recovery in a Mouse Model of Japanese Encephalitis

Japanese encephalitis is a severe central nervous system (CNS) inflammatory disease caused by the mosquito-borne flavivirus, Japanese encephalitis virus (JEV). In the current study we have investigated the immune responses against JEV in mice lacking expression of the chemokine receptor CCR5, which functions in activation and chemotaxis of leukocytes during infection. We show that CCR5 serves as a host antiviral factor against Japanese encephalitis, with CCR5 deficiency markedly increasing mortality, and viral burden in the CNS. Humoral immune responses, which are essential in recovery from JEV infection, were of similar magnitude in CCR5 sufficient and deficient mice. However, absence of CCR5 resulted in a multifaceted deficiency of cellular immune responses characterized by reduced natural killer and CD8⁺ T cell activity, low splenic cellularity, and impaired trafficking of leukocytes to the brain. Interestingly, adoptive transfer of immune spleen cells, depleted of B lymphocytes, increased resistance of CCR5-deficient recipient mice against JEV regardless of whether the cells were obtained from CCR5-deficient or wild-type donor mice, and only when transferred at one but not at three days post-challenge. This result is consistent with a mechanism by which CCR5 expression enhances lymphocyte activation and thereby promotes host survival in Japanese encephalitis.     

Immunopathology of flavivirus infections

With the recent emergence of the flavivirus, West Nile virus (WNV), in particular, the New York strain of Lineage I WNV in North America in 1999, there has been a significant increase in activity in neurotropic flavivirus research. These viruses cause encephalitis that can result in permanent neurological sequelae or death. Attempts to develop vaccines have made progress, but have been variably successful, despite considerable commercial underwriting. Thus, the discovery of ways and means to combat disease is no less urgent. As such, most recent work has been directed towards dissecting and understanding the pathogenesis of disease, as a way of informing possible approaches to abrogation or amelioration of illness. Whether inherent to flaviviruses or because humans are incidental, dead-end hosts, it is clear that these viruses interact with their human hosts in extremely complex ways. This occurs from the cellular level, at which infection must be established to produce disease, to its interaction with the adaptive immune response, which may result in its eradication, with or without immunopathological and consequent neurological sequelae. As human proximity to and contact with flavivirus insect vectors and amplifying hosts cannot practically be eliminated, our understanding of the pathogenesis of flavivirus-induced diseases, especially with regard to possible targets for treatment, is imperative.     

Inhibition of japanese encephalitis virus infection by flavivirus recombinant e protein domain III

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus closely related to the human pathogens including yellow fever virus, dengue virus and West Nile virus. There are currently no effective antiviral therapies for all of the flavivirus and only a few highly effective vaccines are licensed for human use. In this paper, the E protein domain III (DIII) of six heterologous flaviviruses (DENV1-4, WNV and JEV) was expressed in Escherichia coli successfully. The proteins were purified after a solubilization and refolding procedure, characterized by SDS-PAGE and Western blotting. Competitive inhibition showed that all recombinant flavivirus DIII proteins blocked the entry of JEV into BHK-21 cells. Further studies indicated that antibodies induced by the soluble recombinant flavivirus DIII partially protected mice against lethal JEV challenge. These results demonstrated that recombinant flavivirus DIII proteins could inhibit JEV infection competitively, and immunization with proper folding flavivirus DIII induced cross-protection against JEV infection in mice, implying a possible role of DIII for the cross-protection among flavivirus as well as its use in antigens for immunization in animal models.     

Structural basis of a flavivirus recognized by its neutralizing antibody: solution structure of the domain III of the Japanese encephalitis virus envelope protein

The flavivirus envelope protein is the dominant antigen in eliciting neutralizing antibodies and plays an important role in inducing immunologic responses in the infected host. We have determined the solution structure of the major antigenic domain (domain III) of the Japanese encephalitis virus (JEV) envelope protein. The JEV domain III forms a beta-barrel type structure composed of six antiparallel beta-strands resembling the immunoglobulin constant domain. We have also identified epitopes of the JEV domain III to its neutralizing antibody by chemical shift perturbation measurements. Site-directed mutagenesis experiments are performed to confirm the NMR results. Our study provides a structural basis for understanding the mechanism of immunologic protection and for rational design of vaccines effective against flaviviruses.     

Exocytosis and Fas mediated cytolytic mechanisms exert protection from West Nile virus induced encephalitis in mice

Infection of mice with the flaviviruses West Nile virus (WNV) and Murray Valley encephalitis (MVE) induces cytolytic T-cell responses which are highly cross-reactive on target cells infected with heterologous flaviviruses. Of C57BL/6 mice infected with low doses (10(2)-10(6) PFU) of either virus, 30-40% develop encephalitis and die within 10-12 days. Mice with defects in the Fas or granule exocytosis (perforin and granzymes A and B) pathway of cellular cytotoxicity display reduced mortality and increased survival time when infected with MVE and are protected from encephalitis when deficient in both pathways. This contrasts with infection with WNV where defects in these cytolytic mechanisms increase the percentage of mice that succumb to encephalitis. Thus, no generalizations as to protective or detrimental effects of cytolytic effector functions in recovery from closely related flavivirus infections can be made. Virus-host immune interactions have to be assessed individually and cannot be generalized.     

G Protein-Coupled Receptor Kinase 2 Promotes Flaviviridae Entry and Replication

Flaviviruses cause a wide range of severe diseases ranging from encephalitis to hemorrhagic fever. Discovery of host factors that regulate the fate of flaviviruses in infected cells could provide insight into the molecular mechanisms of infection and therefore facilitate the development of anti-flaviviral drugs. We performed genome-scale siRNA screens to discover human host factors required for yellow fever virus (YFV) propagation. Using a 2×2 siRNA pool screening format and a duplicate of the screen, we identified a high confidence list of YFV host factors. To find commonalities between flaviviruses, these candidates were compared to host factors previously identified for West Nile virus (WNV) and dengue virus (DENV). This comparison highlighted a potential requirement for the G protein-coupled receptor kinase family, GRKs, for flaviviral infection. The YFV host candidate GRK2 (also known as ADRBK1) was validated both in siRNA-mediated knockdown HuH-7 cells and in GRK^−/− mouse embryonic fibroblasts. Additionally, we showed that GRK2 was required for efficient propagation of DENV and Hepatitis C virus (HCV) indicating that GRK2 requirement is conserved throughout the Flaviviridae. Finally, we found that GRK2 participates in multiple distinct steps of the flavivirus life cycle by promoting both entry and RNA synthesis. Together, our findings identified GRK2 as a novel regulator of flavivirus infection and suggest that inhibition of GRK2 function may constitute a new approach for treatment of flavivirus associated diseases.     

Lack of both Fas ligand and perforin protects from flavivirus-mediated encephalitis in mice

The mechanism by which encephalitic flaviviruses enter the brain to inflict a life-threatening encephalomyelitis in a small percentage of infected individuals is obscure. We investigated this issue in a mouse model for flavivirus encephalitis in which the virus was administered to 6-week-old animals by the intravenous route, analogous to the portal of entry in natural infections, using a virus dose in the range experienced following the bite of an infectious mosquito. In this model, infection with 0.1 to 10(5) PFU of virus gave mortality in approximately 50% of animals despite low or undetectable virus growth in extraneural tissues. We show that the cytolytic effector functions play a crucial role in invasion of the encephalitic flavivirus into the brain. Mice deficient in either the granule exocytosis- or Fas-mediated pathway of cytotoxicity showed delayed and reduced mortality. Mice deficient in both cytotoxic effector functions were resistant to a low-dose peripheral infection with the neurotropic virus.     

Salicylates inhibit flavivirus replication independently of blocking nuclear factor kappa B activation

Flaviviruses comprise a positive-sense RNA genome that replicates exclusively in the cytoplasm of infected cells. Whether flaviviruses require an activated nuclear factor(s) to complete their life cycle and trigger apoptosis in infected cells remains elusive. Flavivirus infections quickly activate nuclear factor kappa B (NF-kappaB), and salicylates have been shown to inhibit NF-kappaB activation. In this study, we investigated whether salicylates suppress flavivirus replication and virus-induced apoptosis in cultured cells. In a dose-dependent inhibition, we found salicylates within a range of 1 to 5 mM not only restricted flavivirus replication but also abrogated flavivirus-triggered apoptosis. However, flavivirus replication was not affected by a specific NF-kappaB peptide inhibitor, SN50, and a proteosome inhibitor, lactacystin. Flaviviruses also replicated and triggered apoptosis in cells stably expressing IkappaBalpha-DeltaN, a dominant-negative mutant that antagonizes NF-kappaB activation, as readily as in wild-type BHK-21 cells, suggesting that NF-kappaB activation is not essential for either flavivirus replication or flavivirus-induced apoptosis. Salicylates still diminished flavivirus replication and blocked apoptosis in the same IkappaBalpha-DeltaN cells. This inhibition of flaviviruses by salicylates could be partially reversed by a specific p38 mitogen-activated protein (MAP) kinase inhibitor, SB203580. Together, these results show that the mechanism by which salicylates suppress flavivirus infection may involve p38 MAP kinase activity but is independent of blocking the NF-kappaB pathway.     

黄病毒基因组非编码区的结构与功能研究进展

黄病毒科黄病毒属是由一组含单股正链RNA基因组的囊膜病毒组成,包括登革病毒、流行性乙型脑炎病毒、寨卡病毒、西尼罗病毒、黄热病病毒等,经由虫媒传播,可引起人类和动物的严重虫媒病毒病。黄病毒基因组由1个开放阅读框、5′非编码区和3′非编码区三部分组成。5′和3′非编码区含有病毒基因组复制所必需的启动元件;高度结构化的3′非编码区负责黄病毒亚基因组RNA的产生,从而有助于病毒逃避宿主免疫反应;此外3′非编码区还可作为疫苗研究的靶标。鉴于非编码区在黄病毒的蛋白翻译、基因组复制和免疫调节中发挥的重要作用,本文就黄病毒基因组非编码区的结构与功能最新研究进展作一简要综述。     

Flaviviruses

Family Flaviviridae genus flavivirus contains numerous pathogenic viruses such as Japanese encephalitis virus, dengue virus, West Nile virus, etc, which cause public health problems in the world. Since many mammals and birds can act as amplifying hosts and reservoir hosts in nature and those viruses are transmitted by haematophagous mosquitoes or ticks, those viruses could not be eradicated from the nature. In the recent few decades, the viral replication mechanism and the ultrastructure of viral proteins as well as the viral immune evasion mechanism have been elucidated extensively, leading to develop novel types of antivirals and vaccines. In this review, the flavivirus nature and epidemiology, replication mechanism, immune response and immune evasion, and antivirals and vaccines against flaviviruses were described.     

Flavivirus recruits the valosin-containing protein–NPL4 complex to induce stress granule disassembly for efficient viral genome replication

Flaviviruses are human pathogens that can cause severe diseases, such as dengue fever and Japanese encephalitis, which can lead to death. Valosin-containing protein (VCP)/p97, a cellular ATPase associated with diverse cellular activities (AAA-ATPase), is reported to have multiple roles in flavivirus replication. Nevertheless, the importance of each role still has not been addressed. In this study, the functions of 17 VCP mutants that are reportedly unable to interact with the VCP cofactors were validated using the short-interfering RNA rescue experiments. Our findings of this study suggested that VCP exerts its functions in replication of the Japanese encephalitis virus by interacting with the VCP cofactor nuclear protein localization 4 (NPL4). We show that the depletion of NPL4 impaired the early stage of viral genome replication. In addition, we demonstrate that the direct interaction between NPL4 and viral nonstructural protein (NS4B) is critical for the translocation of NS4B to the sites of viral replication. Finally, we found that Japanese encephalitis virus and dengue virus promoted stress granule formation only in VCP inhibitor-treated cells and the expression of NS4B or VCP attenuated stress granule formation mediated by protein kinase R, which is generally known to be activated by type I interferon and viral genome RNA. These results suggest that the NS4B-mediated recruitment of VCP to the virus replication site inhibits cellular stress responses and consequently facilitates viral protein synthesis in the flavivirus-infected cells.